Detection of Bacterial and Phytoplasmal Pathogens in the Environment

Abstract

Plant pathogenic bacteria, except a few, can exist outside plant hosts for short or long periods in soil, water or air and infect the healthy susceptible plants, when they are available. Various methods of detection of bacterial pathogens in the environmental samples have been followed. Biological methods depending on their ability to develop in cell-free media and pathogenicity are the basic and essential methods, although they may be time-consuming and labor intensive. Immunological assays and nucleic acid-based techniques provide more sensitive and specific detection and quantification of pathogen population in the environment especially in the soil samples. These methods are useful in establishing the identity of bacterial pathogens conclusively, when many other saprophytic bacteria are present. Diagnostic methods are very useful in assessing the host range of bacterial pathogens and this information will be required for epidemiological studies.

Phytoplasmas, the cell wall-less bacteria-like organisms require living cells for their existence. They can infect other plant species and natural insect vectors. Both may be able to serve as sources of infection of the primary crop hosts. Various diagnostic techniques especially nucleic acid-based procedures have been applied for the detection, quantification and differentiation as well as for the classification of phytoplasmas in the plant hosts and also in the vector insects. Concerted attempts of researchers have recently succeeded in culturing one of the phytoplasmas in cell-free media, opening up the possibility of rapid advancement in understanding the biology of these pathogens and consequent development of effective management methods for the phytoplasmal diseases.

Bacterial plant pathogens can exist, outside plant hosts, in the soil, water and air. On the other hand, phytoplasmal pathogens are obligate pathogens and hence, they are incapable of existing outside the host. Both bacterial and phytoplasmal pathogens exhibit biological relationships with various insect species that carry them even to long distances breaking the geographical barriers and making the efforts of various countries to contain the spread of these pathogens ineffective. However, consistent attempts have been made to detect these pathogens in different habitats to develop suitable disease management systems.

Appendices

Appendix 1: Detection of Ralstonia solanacearum Race 1 Strains in Soils by BIO-PCR Assay (Lin et al. 2009)

3.1.1 Bacterial Enrichment Using MSM-1 Broth

• 1. Prepare the MSM-1 medium without agar consisting of 10 g peptone, 5 g glucose, 1 g casein hydrolysate, 50 mg 2,3,5-triphenyl tetrazolium chloride (TTC) in 1 l sterile distilled water (SDW) (deleting 15 g agar) with additional antimicrobial compound – 5 mg chloramphenicol, 5 mg crystal violet, 5 mg cycloheximide, 100 mg polymyxin B sulphate and 20 mg tyrothricin.

• 2. Transfer 10 g soil sample to 90 ml SDW; shake at 180 rpm at room temperature for 30 min; add 1 ml sample suspension to 9 ml MSM-1 broth and incubate at 30°C at 160 rpm for enrichment.

• 3. Transfer 5 μl enriched suspension to 200 μl PCR tube; cover with one drop of sterile mineral oil; boil for 5 min and keep the tube in ice.

• 4. Perform the PCR amplification using primer pair AU759f/AU760r with a 25-μl reaction mixture [consisting of 1 × PCR buffer containing 10 mM Tris-HCl, pH 9.0, 50 mM KCl and 0.1% TritonX-100)], 1.5 mM MgCl₂, 0.005 mM each dNTP, 1 pmol of each primer, 2 U Taq DNA polymerase (Promega, USA) and 5 μl boiled bacterial suspension or enriched cultures.

• 5. Provide the following conditions using a thermal cycler: denaturing at 94°C for 3 min, annealing at 53°C for 1 min, extension at 72°C for 1.5 min followed by 30 cycles of 94°C for 18 s, 60°C for 18 s and 72°C for 5 min.

• 6. Resolve the PCR product of 282-bp on 1.5% agarose gel and staining in ethidium bromide solution (1 μg/ml) and visualize under UV light.

Appendix 2: Detection of Xylella fastidiosa (Xf) in Vector Insects by PCR Assay (Krell et al. 2007)

3.2.1 Extraction of DNA

• 1. Use DNeasy Tissue Kit (Qiagen, USA) to extract DNA separately from heads of Homalodisca liturata (Hl) and heads, thoraxes and abdomens of Diceroprocta apache (Da) and grind the individual insects in phosphate buffered saline (PBS) in sample extraction pouches (Agadia).

• 2. Use Ready-To-Go PCR beads (Amersham Biosciences, UK) with a grape-specific primer pair XF2542-L and XF2542-R for PCR amplifications; for each reaction provide one Ready-To-Go PCR bead, 0.25 μl of each primer, 10 μl extracted DNA and 14.5 μl sterile water.

• 3. Perform PCR amplification using a thermal cycler programmed for one cycle of 95°C for 5 min followed by 35 cycles of 94°C for 40 s, 55°C for 40 s, 72°C for 1 min and finally one cycle of 72°C for 5 min and held at 4°C.

• 4. Resolve the amplicons by electrophoresis on 1.5% agarose-tris-borate-EDTA gel and stained with ethidium bromide (0.5 μg/ml of gel) to visualize the separated bands.